U.S. patent application number 14/293397 was filed with the patent office on 2014-12-04 for hydrogel-mediated tissue analysis.
This patent application is currently assigned to Vanderbilt University. The applicant listed for this patent is Vanderbilt University. Invention is credited to Richard Caprioli, Glenn A. Harris, Joshua J. Nicklay.
Application Number | 20140357526 14/293397 |
Document ID | / |
Family ID | 51985795 |
Filed Date | 2014-12-04 |
United States Patent
Application |
20140357526 |
Kind Code |
A1 |
Caprioli; Richard ; et
al. |
December 4, 2014 |
HYDROGEL-MEDIATED TISSUE ANALYSIS
Abstract
A method for analyzing the polypeptide content of animal tissue
is described. The method includes the steps of (a) providing an
animal tissue specimen; (b) depositing one or more portions of a
hydrogel mixture including a protease on spatially discrete
portions of the animal tissue specimen; (c) allowing sufficient
time to pass for animal tissue under the hydrogel mixture to be
form a digested mixture of animal tissue and hydrogel mixture; (d)
removing the digested mixture from the animal tissue and extracting
the polypeptides from the digested mixture to provide an extract;
and (e) analyzing the polypeptide content of the extract by mass
spectrometry.
Inventors: |
Caprioli; Richard;
(Brentwood, TN) ; Harris; Glenn A.; (Nashville,
TX) ; Nicklay; Joshua J.; (Nashville, TN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vanderbilt University |
Nashville |
TN |
US |
|
|
Assignee: |
Vanderbilt University
Nashville
TN
|
Family ID: |
51985795 |
Appl. No.: |
14/293397 |
Filed: |
June 2, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61829524 |
May 31, 2013 |
|
|
|
Current U.S.
Class: |
506/12 ; 435/15;
435/19; 435/21; 435/23 |
Current CPC
Class: |
G01N 2570/00 20130101;
G01N 33/6851 20130101; G01N 33/6887 20130101; C12Q 1/37 20130101;
G01N 33/6848 20130101 |
Class at
Publication: |
506/12 ; 435/23;
435/15; 435/21; 435/19 |
International
Class: |
G01N 33/68 20060101
G01N033/68; C12Q 1/37 20060101 C12Q001/37 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under grant
5P41RR031461-02 awarded by the National Center for Research
Resources and under grant 8 P41 GM103391-02 awarded by the National
Institute of General Medical Sciences from the National Institutes
of Health. The Government has certain rights in this invention.
Claims
1. A method for analyzing the polypeptide content of animal tissue,
comprising: (a) providing an animal tissue specimen; (b) depositing
one or more portions of a hydrogel mixture including a protease on
spatially discrete portions of the animal tissue specimen; (c)
allowing sufficient time to pass for animal tissue under the
hydrogel mixture to form a digested mixture of animal tissue and
hydrogel mixture; (d) removing the digested mixture from the animal
tissue and extracting polypeptides from the digested mixture to
provide an extract; and (e) analyzing the polypeptide content of
the extract by mass spectrometry.
2. The method of claim 1, wherein a plurality of portions of
hydrogel mixture are deposited to provide a plurality of extracts
that are analyzed by mass spectrometry.
3. The method of claim 2, wherein the polypeptide content from a
plurality of spatially discrete regions of the animal tissue
specimen are compared.
4. The method of claim 1, wherein the animal tissue is diseased or
injured.
5. The method of claim 1, wherein the animal tissue is heart
tissue, liver tissue, kidney tissue, prostate tissue, breast
tissue, ovary tissue, uterine tissue, skin tissue, lung tissue,
brain tissue, colon tissue, pancreatic tissue, or muscle
tissue.
6. The method of claim 1, wherein the hydrogel is an ionotropic
hydrogel.
7. The method of claim 1, wherein the hydrogel comprises an
alginate or polyacrylamide polymer.
8. The method of claim 1, wherein the protease is trypsin.
9. The method of claim 1, wherein the animal tissue specimen has a
thickness ranging from about 5 .mu.m to about 50 .mu.m.
10. The method of claim 1, wherein the hydrogel portions are drops
or discs.
11. The method of claim 10, wherein the drops or discs have a
diameter ranging from about 250 .mu.m to about 1 mm.
12. The method of claim 1, wherein the hydrogel portions are
prepared using a paper template.
13. The method of claim 1, wherein the mass spectrometry is MALDI
mass spectrometry or LC-MS/MS.
14. The method of claim 1, wherein the hydrogel mixture is heated
during formation of the digested mixture.
15. The method of claim 13, wherein the hydrogel mixture is heated
using microwave radiation.
16. The method of claim 1, wherein one or more steps of the method
are automated.
Description
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 61/829,524, filed May 31, 2013, the disclosure
of which is incorporated by reference herein.
BACKGROUND
[0003] Tissue analyses, including histomorphological and
immunohistochemical approaches, form the basis for most diagnostic
analyses in anatomic pathology. Highly standardized approaches and
rigorous training regimens have been instituted to ensure that
these morphological approaches to disease characterization deliver
a high standard of care; therefore, many patient lives are saved
annually using these approaches. However, in spite of these
safeguards, there still exist situations for which the current
methods do not provide a definitive diagnosis. For these
unfortunate cases, new technological approaches that incorporate
molecular analysis would add significant value to the diagnostic
process. The development of proteomics and mass spectrometry
technologies during the previous decade has enabled rapid and
specific protein analyses. These technical advances now provide the
opportunity to contribute molecular information with high chemical
and spatial specificity at sufficient throughput to aid in the
histopathological evaluation of patient specimens.
[0004] Protein analysis and identification are traditionally
performed through the use of one of two different strategies. Using
gel electrophoresis, proteins are separated in one or two
dimensions (1D/2D) on a gel and enzymatic digestion is performed
in-gel, a time-consuming and manual process. In a second
solution-based approach, proteins or peptides are separated
chromatographically using on-line LC systems and the proteins are
digested in solution prior to the chromatographic analysis. Capelo
et al., Anal. Chim. Acta 650, 151-9 (2009). The in-solution
approach tends to be the simplest in terms of sample handling and
speed, but the digestion step is still the most time-consuming step
in the sample preparation workflow. Another disadvantage to this
approach is the requirement for sample homogenization. Common
proteomics workflows such as those described require microgram to
milligram quantities of proteins be extracted from the tissue to
provide sufficient material to perform the analysis. This requires
the homogenization of the bulk sample, a step that can
significantly diminish the possibility of studying specific cells
in relation to their native environment in the tissue.
[0005] Histology-guided approaches to the sampling or analysis of
tissues have been developed that can overcome these problems. For
example, groups have reported the use of laser microdissection (LM)
to sample specific cell types from tissues (both fresh and formalin
fixed) (Mukherjee et al., Methods in Mol. Biol. 1002, 71-83 (2013))
and subsequent analysis of these samples using a variety of
genomics and proteomics approaches. This approach has been utilized
to study the molecular content in histologically distinct tissue
regions in a variety of disease states. Jain et al., Am. J. of
Kidney Disease 63, 324-328 (2014); Xing et al., Oncology Reports
31, 634-640 (2014). Furthermore, there now exists a
proteomics-based diagnostic test that combine LM with LC-MS/MS to
type specific amyloid proteins in patient biopsies. Vrana et al.,
Blood 114, 4957-9 (2009). In spite of the advantages and the
utility of LM as a sampling approach for proteomics of tissue
specimens, throughput is very limited, making it difficult to
anticipate analyzing more than a few samples at one time.
[0006] One alternative is to perform the digestion directly on
cryosectioned tissue, then identify the proteins or their
constituent peptides directly from the tissue surface via tandem MS
(MS/MS) and accurate mass measurements. The bottom-up approach,
including in situ proteolytic digestion, is often used to identify
a pool of proteins from which all potential biomarkers are most
likely derived. Groseclose et al., J Mass Spectrom., 42(2), 254-62
(2007). However, on-tissue protein identification can be laborious.
Many traditional proteomic methodologies to identify proteins may
involve one of several approaches such as microextraction with
solvents from the tissue surface, tissue homogenization using
multiple tissue specimens or laser capture microdissection (LCM) of
the regions of interest on a single tissue specimen. Franck et al.,
Mol. Cell. Proteomics 8, 2023-33 (2009); Xu et al., J. Am. Soc.
Mass Spectrom. 13, 1292-7 (2002). All of these approaches require
an overnight digestion, an approach that can be problematic for
analyses of such small volumes on-tissue surfaces where evaporation
and delocalization of the solvent can stop the digestion
prematurely.
[0007] The enzymatic digestion step is commonly the bottleneck of
the workflows used in proteomics. Previously, many research teams
have developed new protocols for protein digestion and
identification that are designed to reduce the sample handling
while increasing sample throughput. Santos et al., Proteomics 13,
1423-7 (2013). These two goals have been achieved by reducing the
total time of the entire workflow or increasing the number of
samples treated at the same time. Many tools have been successfully
used to accelerate the enzymatic digestion of proteins: for
example, heating, microspin columns, ultrasonic energy, high
pressure, infrared energy, alternating electric field or microwave.
Juan et al., Proteomics 5, 840-2 (2005). While microwave assisted
proteolytic digestion has traditionally been implemented in
solution, there is a growing trend to use heterogeneous systems for
on-tissue digestion in which enzyme is carried within hydrogels or
adsorbed on solid supports. Fiddes et al., Biomicrofluidics 6,
14112-1411211 (2012); Luk et al., Proteomics 12, 1310-8 (2012).
Molecular hydrogels have attracted extensive research interest
recently because of their great potential for tissue engineering,
migration of organic and inorganic material, drug delivery as well
as a miniaturized method for application on biological samples.
Toledano et al., J. Am. Chem. Soc. 128, 1070-1 (2006); Li et al.,
Chem. Commun. (Carob). 48, 6175-7 (2012).
[0008] Improvements and complementary methods are needed to address
difficulties and challenges of the on-tissue identification
process. More user-friendly approaches should be adopted to obviate
the need of costly robotic liquid extraction, matrix deposition and
tissue isolating instruments.
SUMMARY
[0009] Proteomics is an extremely powerful tool in examining
cellular function, and provides a complementary analysis to
genomics efforts. While it is somewhat more complicated to examine
protein expression profiles, mass spectrometry (MS), because of its
extreme selectivity and sensitivity, has now become a favored tool
in the global examination of protein expression. However, a
limitation on any analysis of this nature is the need to
interrogate molecular changes in discrete tissue samples while
permitting high throughput.
[0010] Traditional methods of examining proteomes with MS involve
homogenizing small samples of tissues, using separative techniques
such as 2D gels or liquid chromatography, which are followed by MS
for detection. Although this method gives adequate results, it is
tedious, labor intensive and destroys any spatial fidelity in the
sample due to the homogenization process. Therefore, current
approaches to MS quantification of protein expression require
substantial improvements in sample processing and utilization.
[0011] The present inventors have developed a method for analyzing
protein expression in situ, i.e., directly in intact tissues and
within discrete areas thereof. In particular embodiments, one or
more portions of a hydrogel mixture including a protease are
deposited on spatially discrete portions of an animal tissue
specimen. The protease of the hydrogel mixture results in the
formation of a digested mixture of animal tissue and hydrogel
mixture. The digested mixture is then removed from the animal
tissue and polypeptides are extracted to provide an extract. The
polypeptide content of the extract is then analyzed by mass
spectrometry.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1 provides a scheme showing hydrogel-mediated proteomic
digestion and extraction workflow beginning with a) hydrogel
synthesis on a laser printed piece of chromatography paper, b)
on-tissue placement of gel for proteolysis and incubation, c)
solvent extraction in aqueous and organic solvents, and analysis of
reconstituted extracts with d) MALDI MS and/or e) LC-MS/MS.
[0013] FIG. 2 provides mass spec. images showing MALDI MS profile
analysis of 2% (by volume) of the reconstituted hydrogel mediated
digestion extracts from rat brain cerebrum (12 .mu.m thickness).
Linear mode full scan spectra (m/z 1500-10000, 2000 shots summed)
from a) trypsin loaded hydrogel placed off-tissue (blank), b)
on-tissue hydrogel without trypsin (control) and c) an on-tissue
trypsin hydrogel (digest).
[0014] FIG. 3 provides images showing a) Image overlay of three PC
lipid species analyzed using ion mobility MALDI IMS:PC 36:1, PC
34:1, and PC 36:4. Scale bars are 2 mm. On-tissue MS/MS spectra for
the precursor b) protonated ion of PC 36:1 at m/z 788.62, c)
potassiated ion of PC 34:1 at m/z 798.54 and d) sodiated ion PC
36:4 at m/z 804.55 were obtained prior to on-tissue hydrogel
digestion.
[0015] FIG. 4 provides images showing a) hematoxylin and eosin (H
& E) stain of an untreated rat cerebellum region with outlined
(circle) region of where the hydrogel was placed on the serial
digested tissue (scale bar 250 .mu.m) and b) the H & E stain of
the serial sectioned, previously imaged (FIG. 3) and hydrogel
digested rat cerebellum (scale bar 50 .mu.m).
[0016] FIG. 5 provides a scheme showing the workflow of hydrogel
discs fabrication and of the on-tissue digestion is here presented.
a) a bis-polyacrylamide solution is prepared and allowed to
polymerize for 20 min. Once the gel is formed in a glass petri dish
(5 cm diameter), hydrogel discs are created using a punch biopsy
tool at 1 mm diameter. About 150 hydrogel discs are created per 5
cm dish. The discs are then placed into separate eppendorf tubes,
fully dried and stored at 4.degree. C. until use. To start the
digestion experiment, a hydrogel disc is swelled in few microliters
of the enzyme solution and then placed on the region of interest
within the tissue specimen. b) the tissue specimen with the
hydrogel disc on the surface is heated in the microwave oven for 2
min to allow the digestion. After that, the disc is removed from
the tissue surface and placed into an eppendorf tube. A solvent
extraction is carried out and, once dried, the solution is
reconstituted as described herein and ready to be used for both
MALDI MS profiling as well as for LC-MS/MS analysis followed by
data base search for protein identification.
[0017] FIG. 6 provides a scheme showing a workflow of the
histology-directed on-tissue enzymatic digestion; a) H&E of a
fresh frozen rat brain tissue specimen (cryosectioned at 8 .mu.m),
stained for histological evaluation and localization of the brain
thalamic region. Enzymatic digestions were performed depositing the
hydrogel disc embedded with trypsin on the thalamic region and then
incubating the tissue specimen into the microwave for 2 min;
further, a consecutive cut tissue specimen was incubated in an
oven; b) the rat brain punch biopsy was obtained from the thalamic
region at the same diameter of the hydrogel disc (1 mm) and then
cryosectioned at 8 .mu.m. Protein digestion experiments were
carried out using the hydrogel device as well as manual spotting
the enzyme solution and homogenizing tissue specimens within a
conventional oven.
[0018] FIG. 7 provides a MALDI MS spectra of the solvent extracted
digested peptides obtained from the first two hydrogel experiments,
carried out via microwave (grey) and oven (black). The resulting
profiles display a high degree similarity in the ions present in
the mass range 500-4000 Da.
[0019] FIG. 8 provides a MALDI MS spectra of the three technical
replicates of microwave assisted hydrogel mediated on-tissue
digested extracts. The resulting profiles display a high degree
similarity in both the ions present and their relative abundance in
the mass range 500-4000 Da.
[0020] FIG. 9 provides a Venn diagrams summarizing the number of
identified proteins for N=3 on-tissue digestion experiments (5%
FDR, .gtoreq.2 unique peptides and p value <0.05). a) shows the
number of identified proteins within the microwave digested
extracts and the oven incubation digested extracts, both carried
out using the hydrogel disc placed onto the rat brain thalamic
region; b) displays the number of identified proteins within the
experiments performed using the 1 mm diameter tissue specimen from
the same region of interest within the rat brain biopsy, using the
hydrogel disc and homogenizing the tissue specimens
respectively.
[0021] FIG. 10 provides bar graphs shows the number of protein
groups and of distinct peptides obtained from the hydrogel
experiments carried out via microwave and oven (a) and from the
experiments performed on the 1 mm diameter rat brain tissue
specimens from thalamic region (h). The results are expressed as
mean.+-.SD (N=3). Data are averaged from N=3 replicated experiments
per class. Asterisk denotes comparisons found to be statistically
significant, p<0.05.
DETAILED DESCRIPTION
[0022] A method for analyzing the polypeptide content of animal
tissue is described. The method includes the steps of (a) providing
an animal tissue specimen; (b) depositing one or more portions of a
hydrogel mixture including a protease on spatially discrete
portions of the animal tissue specimen; (c) allowing sufficient
time to pass for animal tissue under the hydrogel mixture to be
form a digested mixture of animal tissue and hydrogel mixture; (d)
removing the digested mixture from the animal tissue and extracting
the polypeptides from the digested mixture to provide an extract;
and (e) analyzing the polypeptide content of the extract by mass
spectrometry.
DEFINITIONS
[0023] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains. In case
of conflict, the present specification, including definitions, will
control.
[0024] The terminology as set forth herein is for description of
the embodiments only and should not be construed as limiting the
application as a whole. Unless otherwise specified, "a," "an,"
"the," and "at least one" are used interchangeably. Furthermore, as
used in the description of the application and the appended claims,
the singular forms "a", "an", and "the" are inclusive of their
plural forms, unless contraindicated by the context surrounding
such. Furthermore, the recitation of numerical ranges by endpoints
includes all of the numbers subsumed within that range (e.g., 1 to
5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
[0025] As used herein, the term "polypeptide" refers to an
oligopeptide, peptide, or protein, or to a fragment, portion, or
subunit of any of these, and to naturally occurring or synthetic
molecules. The term "polypeptide" also includes amino acids joined
to each other by peptide bonds or modified peptide bonds, i.e.,
peptide isosteres, and may contain any type of modified amino
acids. The term "polypeptide" also includes peptides and
polypeptide fragments, motifs and the like, glycosylated
polypeptides, all "mimetic" and "peptidomimetic" polypeptide forms,
and retro-inversion peptides (also referred to as all-D-retro or
retro-enantio peptides). Generally, a peptide has less than 30
amino acids, whereas a protein has more than 30 amino acids, though
this is an approximate dividing line between the two.
[0026] A subject can be any animal, and animal tissue described
herein can be obtained from any type of subject. In some
embodiments, the subject is a mammal, such as a domesticated farm
animal (e.g., cow, horse, pig) or pet (e.g., dog, cat). More
preferably, the subject is a human. When the subject is a human,
the subject may also be referred to as a patient, particularly when
the subject is being evaluated in a medical environment or by
medical personnel.
[0027] In one aspect, the present invention provides a method for
analyzing the polypeptide content of animal tissue. The method
includes the steps of (a) providing an animal tissue specimen; (b)
depositing one or more portions of a hydrogel mixture including a
protease on spatially discrete portions of the animal tissue
specimen; (c) allowing sufficient time to pass for animal tissue
under the hydrogel mixture to be form a digested mixture of animal
tissue and hydrogel mixture; (d) removing the digested mixture from
the animal tissue and extracting the polypeptides from the digested
mixture to provide an extract; and (e) analyzing the polypeptide
content of the extract by mass spectrometry.
[0028] The method includes the step of depositing one or more
portions of a hydrogel mixture including a protease onto spatially
discrete portions of the animal tissue specimen. A hydro gel may be
defined as a three-dimensional, hydrophilic or amphiphilic
polymeric network capable of taking up large quantities of water.
The networks are composed of homopolymers or copolymers, are
insoluble due to the presence of covalent chemical or physical
(ionic, hydrophobic interactions, entanglements) crosslinks. The
crosslinks provide the network structure and physical integrity.
Hydrogels exhibit a thermodynamic compatibility with water that
allows them to swell in aqueous media.
[0029] In some embodiments, the hydrogel is prepared by
crosslinking hydrophilic biopolymers or synthetic polymers.
Examples of the hydrogels formed from physical or chemical
crosslinking of hydrophilic biopolymers, include but are not
limited to, hyaluronans, chitosans, alginates, collagen, dextran,
pectin, carrageenan, polylysine, gelatin or agarose. Examples of
hydrogels based on crosslinked synthetic polymers include but are
not limited to
(meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate,
poly(ethylene glycol) (PEO), poly(propylene glycol) (PPO),
PEO-PPO-PEO copolymers (Pluronics), poly(phosphazene),
poly(methacrylates), poly(N-vinylpyrrolidone), PL(G)A-PEO-PL(G)A
copolymers, poly(ethylene imine), etc. See A. S Hoffman, Adv. Drug
Del. Rev 43, 3-12 (2002). In some embodiments, the hydrogel is an
alginate or polyacrylamide hydrogel.
[0030] In some embodiments, the hydrogel is an ionotropic hydrogel.
When a polyelectrolyte is combined with a multivalent ion of the
opposite charge, it may form a physical hydrogel known as an
`ionotropic` hydrogel. Calcium alginate is an example of an
ionotropic hydrogel. Further, when polyelectrolyte of opposite
charges is mixed, they may gel or precipitate depending on their
concentrations, the ionic strength, and pH of the solution. The
products of such ion crosslinked systems are known as complex
coacervates, polyion complexes, or polyelectrolyte complexes.
[0031] In some embodiments, one or more portions of the hydrogel
including the protease are placed directly on the animal tissue
specimen. The portions of hydrogel can vary in size and shape. In
some embodiments, the hydrogel portions are drops or discs. In some
embodiments, the drops or discs have a diameter ranging from about
100 .mu.m to about 5 mm, while in other embodiments, the drops or
discs have a diameter ranging from about 250 .mu.m to about 1 mm,
or from about 400 to 700 .mu.m. The size and shape of the hydrogel
portion determines the size and shape of the animal tissue section
within the specimen that is evaluated using that portion.
[0032] In some embodiments, the hydrogel is first applied in a low
viscosity state to a paper template and thereafter converted into a
high viscosity gel when applied. Use of a paper template
facilitates preparing hydrogel portions in whatever size and shape
is desired. For example, methods of forming hydrogels using paper
are described in International Publication No. WO 2009/121038,
entitled "Shaped Films of Hydrogels Fabricated Using Templates of
Patterned Paper," filed Mar. 27, 2009, incorporated in its entirety
by reference. The paper can be printed with the desired shapes
corresponding to that of the portion of hydrogel that is applied to
the animal tissue specimen. By using hydrophobic ink, hydrophobic
boundaries can be created on the paper surface. A preferred type of
paper is chromatography paper. In one exemplary method, an
ionotropic hydrogel is formed by contacting the substrate with a
solution of one or more gelling agents, including but not limited
to metallic ions, such as Pb.sup.2+, Ba.sup.2+, Fe.sup.3+,
Al.sup.3+, Cu.sup.2+, Cd.sup.2+, Ho.sup.3+, Ca.sup.2+, Zn.sup.2+,
Co.sup.2+, Ni.sup.2+, Mn.sup.2+, and Mg.sup.2+, and contacting the
substrate with a hydrogel precursor such as alginic acid (AA),
carboxymethylcellulose (CMC), -carrageenan, poly(galacturonic acid)
(PG), or acrylamide/bisacrylamide. The interaction of the gelling
agent, e.g., ion, with the hydrogel precursor results in gelation
of the hydrogel.
[0033] Typically, the method of tissue analysis involves obtaining
tissue samples from a variety of locations on the subject or a
particular animal tissue specimen. In such embodiments, a plurality
of portions of hydrogel mixture are deposited to provide a
plurality of extracts for analysis by mass spectrometry. For
example, 5 or more, or 10 or more, or 50 or more portions of
hydrogel mixture can be deposited. The portions of hydrogel mixture
should be deposited on spatially discrete regions of the animal
tissue specimen. A spatially discrete region of animal tissue is a
region that does not overlap with another region that is being
analyzed. In some embodiments, the portions of hydrogel mixture can
be deposited in a regular pattern in order to facilitate conducting
and/or automating the assay. For example, the portions of hydrogel
can be positioned in regularly spaced rows and columns. When
conducting the method of tissue analysis, it can be useful to
compare the polypeptide content from a plurality of spatially
discrete regions of the animal tissue.
Animal Tissue Specimens
[0034] In accordance with the present invention, intact tissue
samples are obtained by standard methodologies for use as animal
tissue specimens. The tissue samples must be of a sufficient size
to permit creation of a plurality of microregions, e.g., at least 1
micron to several millimeters, including sizes in between, such as
2 .mu.m, 3 .mu.m, 4 .mu.m, 5 .mu.m, 6 .mu.m, 7 .mu.m, 8 .mu.m, 9
.mu.m, 10 .mu.m, 15 .mu.m, 20 .mu.m, 25 .mu.m, 30 .mu.m, 35 .mu.m,
40 .mu.m, 45 m, 50 m, 60 .mu.m, 70 .mu.m, 80 .mu.m, 90 .mu.m, 100
.mu.m, 125 .mu.m, 150 .mu.m, 175 .mu.m, 200 .mu.m, 250 .mu.m, 275
.mu.m, 300 .mu.m, 400 .mu.m, 450 tun, 500 .mu.m, 600 .mu.m, 700
.mu.m, 800 .mu.m, 900 .mu.m, 1000 .mu.m, 2 mm, 3 mm, 4 mm and 5 mm.
In some embodiments, the tissue specimen has a thickness ranging
from about 5 .mu.m to about 50 .mu.m. In some embodiments, the
animal tissue specimen is cryosectioned animal tissue, which can be
prepared using a cryostat.
[0035] Any type of animal tissue can be analyzed for its
polypeptide content. The animal tissue evaluated can be healthy
tissue, or it can be tissue that is diseased or injured. Examples
of animal tissue suitable for evaluation using the present
invention include heart tissue, liver tissue, kidney tissue,
prostate tissue, breast tissue, ovary tissue, uterine tissue, skin
tissue, lung tissue, brain tissue, colon tissue, pancreatic tissue,
and muscle tissue.
[0036] Biopsy procedures for obtaining the specimen will generally
involve the sterility required of surgical operations, even though
the tissues being sample are from cadavers or animals that will be
sacrificed. For internal tissues, incisions will be made proximal
to the tissue of interest, followed by retraction, excision of
tissue and surgical closing of the incision. Superficial tissue
sites are accessed by simple excision of the available tissue.
[0037] Tissue specimens should be handled such that (a) the
integrity of the tissue is maintained and (b) that the cells within
the tissue, particularly those in the region(s) where hydrogel
portions will be placed are not damaged. Appropriate physiologic
buffers are generally applied to the tissue, or the tissues are
immersed therein. The tissue may also be cooled to appropriate
temperatures for limited periods of time. Steps should be taken to
ensure that apoptosis or other cellular degradation will not be
induced in the tissue specimen.
[0038] In some embodiments, pretreatment with of tissues prior to
application of the hydrogel may prove advantageous. On particularly
useful pretreatment is an ethanol wash, optionally followed by a
storage period of minutes to hours. In tests on several tissue
types, improved well formation was observed using this approach.
Also, the delivery of matrix in a solvent comprising 10%
acetonitrile, 60% water, 30% isopropanol and 0.5% acetic acid
provided improved results.
Protein Digestion
[0039] A feature of the present invention is that tissue digestion
to release polypeptides (i.e., proteins and/or peptides) from the
tissue is carried out directly on the animal tissue surface (i.e.,
"in situ") using an enzyme included in the hydrogel. Preferably,
the enzyme is a protease (i.e., proteolytic enzyme or proteinase).
Proteases are involved in digesting long protein chains into
shorter fragments by splitting the peptide bonds that link amino
acid residues. Examples of proteases include serine proteases,
threonine proteases, cysteine proteases, aspartate proteases,
glutamic acid proteases, and metalloproteases. Protease digestion
serves to release polypeptides from the animal tissue surface, and
to digest proteins to facilitate analysis my mass spectrometry.
[0040] Most commonly digestions are carried out with the proteases
trypsin or lysine specific proteinases, because these enzymes are
reliable, specific and produce a suitable number of peptides. The
next most common digestion is at aspartate or glutamate using
endoproteinase Glu-C or endoproteinase Asp-N. Chymotrypsin is
sometimes used, although it does not have a well defined
specificity. Proteinases of broad specificity may generate many
peptides, and the peptides may be very short.
[0041] In some embodiments, the hydrogel mixture is heated during
formation of the digested mixture. Heating increases the rate at
which digestion of the tissue occurs, thereby decreasing the amount
of time necessary for a sufficient time to pass for the hydrogel
mixture to form a digested mixture. The hydrogel mixture should be
heated to a temperature above room temperature. For example, in
some embodiments, it is preferable to heat the hydrogel mixture to
a temperature from about 35.degree. C. to about 60.degree. C. for a
time from about 2 to about 12 hours. The heating should be
sufficient to accelerate digestion, without destroying the
protease, polypeptide, or animal tissue. A variety of means can be
used to heat the digested mixture. For example, the digested
mixture can be heated by placing the tissue bearing the hydrogel
mixture into an oven. Preferably the animal tissue is held within
an air-tight container during heating in order to prevent drying of
the hydrogel during heating.
[0042] In some embodiments, the hydrogel mixture is heated using
microwave radiation. Use of microwave radiation has the advantage
of significantly increasing the speed of protein digestion, which
has the effect of further decreasing the amount of time to provide
a sufficient time to pass for the hydrogel mixture to form a
digested mixture. The microwave radiation can be any suitable
wavelength (e.g., from about 0.5 to 5 GHz) and any suitable wattage
(e.g., from about 300 to 3,000 watts). Because of the efficiency of
microwave heating, a sufficient time for digestion can be from 1
minute to 1 hour, with typical settings providing digestion in from
about 1 minute to about 5 minutes.
Extraction and Sample Preparation
[0043] Once one has obtained a digested mixture of animal tissue
and hydrogel, an extraction step is carried out to extract the
polypeptides from the digested mixture in order to provide an
extract for analysis by mass spectrometry. The extraction step can
include an organic extraction and/or an aqueous extraction.
Extraction will shrink and swell the hydrogel in order to release
polypeptides within the digested mixture, and can also separate the
polypeptides, which migrate to the aqueous phase, from other
components in the extraction mixture.
[0044] As the collected samples are to be prepared for proteomic
analysis, proteins should be extracted from lipids, metabolites,
and other non-proteinaceous compounds, which may interfere with
downstream procedures. Various chemical precipitation methods are
available for protein isolation; these include acetone,
trichloroacetic acid (TCA), ethanol, isopropanol,
chloroform/methanol, and ammonium sulfate. The efficiency of
protein precipitation varies among different organic solvents. For
example, acetone has been determined to precipitate more acidic and
hydrophilic proteins, whereas ultracentrifugation fractionates more
basic, hydrophobic, and membrane proteins. Thongboonkerd et al.,
Kidney Int. 62(4):1461-9 (2002). Alternatively, chloroform methanol
extraction has been used to successfully extract hydrophobic
proteins. Stark et al., Eur J. Biochem. 266(1):209-14 (1999).
Precipitation strategies can be optimized for a particular sample
type.
[0045] In some embodiments, it may be preferable to treat the
polypeptide extract with various enzymes (lipases, collagenases,
proteases, nucleases) to further purify the sample. Examples of
these treatments are provided below.
Lipases
[0046] In some embodiments, lipases may be used to further degrade
lipid contaminants. Most lipases exhibit the catalytic triad
Ser-Asp-His (an exception being geotrichium candidum which has
Ser-Glu-His). Lipases have been isolated from a wide variety of
mammalian and microbial sources. The mammalian lipases can be split
into four groups, the hepatic lingual, gastric and pancreatic
lipase and microbial lipases into bacterial and fungal. Very little
homology has been found within the known sequences, the most
conserved feature being the consensus sequence G.times.S.times.G
found in the substrate binding site. The above-mentioned catalytic
triad (Ser-Asp-His) is also a highly conserved. However, this is
common to all esterases, not just lipases, as is the .alpha./.beta.
hydrolase fold.
[0047] Known lipases include triacylglycerol lipase (triglyceride
lipase; tributyrase) phospholipase A2 (phosphatidylcholine
2-acylhydrolase, lecithinase A, phosphatidase, phosphatidolipase)
lysophospholipase (lecithinase B, lysolecithinase, phospholipase B)
acylglycerol lipase (monoacylglycerol lipase) galactolipase,
phospholipase Al, lipoprotein lipase (clearing factor lipase,
diglyceride lipase, diacylglycerol lipase) dihydrocoumarin lipase,
2-acetyl-1-alkylglycerophosphocholine esterase
(1-alkyl-2-acetylglycerophosphocholine esterase,
platelet-activating factor acetylhydrolase, PAP acetylhydrolase,
PAF 2-acylhydrolase, LDL-associated phospholipase A2 LDL-PLA(2)),
phosphatidylinositol deacylase (phosphatidylinositol phospholipase
A2) phospholipase C (lipophosphodiesterase I, Lecithinase C,
Clostridium welchii .alpha.-toxin, Clostridium oedematiens .beta.-
and .gamma.-toxins) phospholipase D, (lipophosphodiesterase II,
lecithinase D, choline phosphatase), phosphoinositide phospholipase
C (triphosphoinositide phosphodiesterase, phosphoinositidase C,
1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase,
monophosphatidylinositol phosphodiesterase. phosphatidylinositol
phospholipase C, PI-PLC,
1-phosphatidyl-D-myo-inositol-4,5-bisphosphate
inositoltrisphosphohydrolase), alkylglycerophosphoethanolamine
phosphodiesterase. (lysophospholipase D),
glycosylphosphatidylinositol phospholipase D (GPI-PLD, glycoprotein
phospholipase D, phosphatidylinositol phospholipase D,
phosphatidylinositol-specific phospholipase D,
phosphatidylinositol-glycan-specific phospholipase D),
phosphatidylinositol diacylglycerol-lyase (1-phosphatidylinositol
phosphodiesterase, monophosphatidylinositol phosphodiesterase,
phosphatidylinositol phospholipase C, 1-phosphatidyl-D-myo-inositol
inositolphosphohydrolase (cyclic-phosphate-forming)),
glycosylphosphatidylinositol diacylglycerol-lyase
((glycosyl)phosphatidylinositol-specific phospholipase C, GPI-PLC,
GPI-specific phospholipase C, VSG-lipase, glycosyl inositol
phospholipid anchor-hydrolyzing enzyme,
glycosylphosphatidylinositol-phospholipase C,
glycosylphosphatidylinositol-specific phospholipase C,
variant-surface-glycoprotein phospholipase C).
Collagenases
[0048] Collagen is the most abundant protein in vertebrates, and
occurs in almost every tissue. However, for many applications, is
it necessary to remove collagen in order to analyze other proteins
in a sample. Moreover, analysis of collagen may be of only limited
interest. As a result, methods for the removal of collagen are
regularly employed. Collagenases are enzymes that are able to
cleave the peptide bonds in triple helical collagen molecules.
Collagenases from Clostridium histolyticum have been known and
studied for decades. Clostridopeptidase and clostripain activities
also are associated with some collagenase preparations.
Nucleic Acid Removal
[0049] Elimination of nucleic acids from sample prior to analysis
can be achieved by chemical or enzymatic means. Chemical removal
involves precipitation methods that employ polyethyleneimine (PEI)
or streptomycin sulfate precipitation, followed by
centrifugation.
[0050] Alternatively, enzymes that specifically degrade DNA and/or
RNA may be used to remove these molecules. Benzonase is a
genetically engineered endonuclease from Serratia marcescens. The
protein is a dimer of two 30 kDa subunits. The enzyme degrades all
forms of DNA and RNA, including single-stranded, double-stranded,
linear and circular molecules, and is effective over a wide range
of operating conditions. Some sequence specificity has been
identified, with GC-rich regions being preferred. More selective
enzymes that degrade DNA (DNases) or RNA (RNases) can be utilized
as well.
Buffers
[0051] Once extracted, buffers will often be utilized to preserve
the integrity of protein samples. Buffers are aqueous composed of a
weak acid (proton donor) and its conjugate base (proton acceptor).
The acid or base is partially neutralized and shows little pH
change in response to the addition of stronger acids or bases
because of the buffers ability to "absorb" hydrogen ions, which
determine pH. The most effective pH range for a buffer is generally
one pH unit and is centered around the pK.sub.a of the system.
[0052] In choosing an appropriate buffer system, one generally
takes into account the following considerations. (1) The pK.sub.a
of the buffer should be near the desired midpoint pH of the
solution. (2) The capacity of the buffer should fall within one to
two pH units above or below the desired pH values. If the pH is
expected to drop during the procedure, choose a buffer with a
pK.sub.a slightly lower than the midpoint pH. Similarly, if the pH
is expected to rise, choose a buffer with a slightly elevated pH.
(3) The concentration of the buffer should be adjusted to have
enough capacity for the experimental system. (4) The pH of the
buffer should be checked at the temperature and concentration which
will be used in the experimental system. (5) No more than 50% of
the buffer components should be dissociated or neutralized by ionic
constituents which are generated within or added to the solution.
(6) Buffer materials should not absorb light between the
wavelengths of 240-700 nm.
[0053] Useful buffers include ADA (Na salt), BES, ethyl glycinate,
glycine, PBS, lithium citrate, PIPPS, potassium phosphate (mono- or
dibasic), sodium citrate, sodium phosphate, TAPS, Tris base,
Tris-HCl, MES, Bis-Tris, PIPES (Na salt), ACES, MOPES, TES, HEPES
(Na salt), HEPPS, Tricine, Bicine, CHES, CAPS, MOPSO, DIPSO,
HEPPSO, POPSO, AMPSO and CAPSO. Particularly useful buffers for
mass spectrometry are volatile buffers, including ammonium
bicarbonate and ammonium acetate.
Mass Spectrometry
[0054] A "mass spectrometer" is an analytical instrument that can
be used to determine the molecular weights of various substances,
such as proteins and nucleic acids. It can also be used in some
applications, e.g., to determine the sequence of protein molecules
and the chemical composition of virtually any material. Typically,
a mass spectrometer comprises four parts: a sample inlet, an
ionization source, a mass analyzer, and a detector. A sample is
optionally introduced via various types of inlets, e.g., solid
probe, GC, or LC, in gas, liquid, or solid phase. The sample is
then typically ionized in the ionization source to form one or more
ions. The resulting ions are introduced into and manipulated by the
mass analyzer. Surviving ions are detected based on mass to charge
ratio. In one embodiment, the mass spectrometer bombards the
substance under investigation with an electron beam and
quantitatively records the result as a spectrum of positive and
negative ion fragments. Separation of the ion fragments is on the
basis of mass to charge ratio of the ions. If all the ions are
singly charged, this separation is essentially based on mass.
Traditional quantitative MS has used electrospray ionization (ESI)
followed by tandem MS (MS/MS) while newer quantitative methods are
being developed using matrix assisted laser desorption/ionization
(MALDI) followed by time of flight (TOF) MS.
A. ESI
[0055] ESI is a convenient ionization technique developed by Fenn
and colleagues (Fenn et al., Science, 246(4926):64-71, 1989) that
is used to produce gaseous ions from highly polar, mostly
nonvolatile biomolecules, including lipids. The sample is injected
as a liquid at low flow rates (1-10 .mu.L/min) through a capillary
tube to which a strong electric field is applied. The field
generates additional charges to the liquid at the end of the
capillary and produces a fine spray of highly charged droplets that
are electrostatically attracted to the mass spectrometer inlet. The
evaporation of the solvent from the surface of a droplet as it
travels through the desolvation chamber increases its charge
density substantially. When this increase exceeds the Rayleigh
stability limit, ions are ejected and ready for MS analysis.
[0056] A typical conventional ESI source consists of a metal
capillary of typically 0.1-0.3 mm in diameter, with a tip held
approximately 0.5 to 5 cm (but more usually 1 to 3 cm) away from an
electrically grounded circular interface having at its center the
sampling orifice. Kabarle et al., Anal. Chem. 65(20):972A-986A
(1993). A potential difference of between 1 to 5 kV (but more
typically 2 to 3 kV) is applied to the capillary by power supply to
generate a high electrostatic field (10.sup.6 to 10.sup.7 V/m) at
the capillary tip. A sample liquid carrying the analyte to be
analyzed by the mass spectrometer, is delivered to tip through an
internal passage from a suitable source (such as from a
chromatograph or directly from a sample solution via a liquid flow
controller). By applying pressure to the sample in the capillary,
the liquid leaves the capillary tip as small highly electrically
charged droplets and further undergoes desolvation and breakdown to
form single or multicharged gas phase ions in the form of an ion
beam. The ions are then collected by the grounded (or negatively
charged) interface plate and led through an orifice into an
analyzer of the mass spectrometer. During this operation, the
voltage applied to the capillary is held constant. Aspects of
construction of ESI sources are described, for example, in U.S.
Pat. Nos. 5,838,002; 5,788,166; 5,757,994; RE 35,413; 6,756,586,
5,572,023 and 5,986,258.
B. ESI/MS/MS
[0057] In ESI tandem mass spectroscopy (ESI/MS/MS), one is able to
simultaneously analyze both precursor ions and product ions,
thereby monitoring a single precursor product reaction and
producing (through selective reaction monitoring (SRM)) a signal
only when the desired precursor ion is present. When the internal
standard is a stable isotope-labeled version of the analyte, this
is known as quantification by the stable isotope dilution method.
This approach has been used to accurately measure pharmaceuticals
(Zweigenbaum et al., Anal. Chem., 74:2446, 2000) and bioactive
peptides (Desiderio et al., Biopolymers, 40:257, 1996). Newer
methods are performed on widely available MALDI-TOF instruments,
which can resolve a wider mass range and have been used to quantify
metabolites, peptides, and proteins. Larger molecules such as
peptides can be quantified using unlabeled homologous peptides as
long as their chemistry is similar to the analyte peptide. Bucknall
et al., J. Am. Soc. Mass Spectrometry, 13(9):1015-27 (2002).
Protein quantification has been achieved by quantifying tryptic
peptides. Mirgorodskaya et al., Rapid Commun. Mass Spectrom.,
14:1226, 2000. Complex mixtures such as crude extracts can be
analyzed, but in some instances sample clean up is required. Gobom
et al., Anal. Chem. 72:3320, 2000. Desporption electrospray is a
new associated technique for sample surface analysis.
C. SIMS
[0058] Secondary ion mass spectroscopy, or SIMS, is an analytical
method that uses ionized particles emitted from a surface for mass
spectroscopy at a sensitivity of detection of a few parts per
billion. The sample surface is bombarded by primary energetic
particles, such as electrons, ions (e.g., O, Cs), neutrals or even
photons, forcing atomic and molecular particles to be ejected from
the surface, a process called sputtering. Since some of these
sputtered particles carry a charge, a mass spectrometer can be used
to measure their mass and charge. Continued sputtering permits
measuring of the exposed elements as material is removed. This in
turn permits one to construct elemental depth profiles. Although
the majority of secondary ionized particles are electrons, it is
the secondary ions which are detected and analysis by the mass
spectrometer in this method.
D. LD-MS and LDLPMS
[0059] Laser desorption mass spectroscopy (LD-MS) involves the use
of a pulsed laser, which induces desorption of sample material from
a sample site--effectively, this means vaporization of sample off
of the sample substrate. This method is usually only used in
conjunction with a mass spectrometer, and can be performed
simultaneously with ionization if one uses the right laser
radiation wavelength.
[0060] When coupled with Time-of-Flight (TOF) measurement, LD-MS is
referred to as LDLPMS (Laser Desorption Laser Photoionization Mass
Spectroscopy). The LDLPMS method of analysis gives instantaneous
volatilization of the sample, and this form of sample fragmentation
permits rapid analysis without any wet extraction chemistry. The
LDLPMS instrumentation provides a profile of the species present
while the retention time is low and the sample size is small. In
LDLPMS, an impactor strip is loaded into a vacuum chamber. The
pulsed laser is fired upon a certain spot of the sample site, and
species present are desorbed and ionized by the laser radiation.
This ionization also causes the molecules to break up into smaller
fragment-ions. The positive or negative ions made are then
accelerated into the flight tube, being detected at the end by a
microchannel plate detector. Signal intensity, or peak height, is
measured as a function of travel time. The applied voltage and
charge of the particular ion determines the kinetic energy, and the
separation of fragments is due to different size causing different
velocity. Each ion mass will thus have a different flight-time to
the detector.
[0061] One can either form positive ions or negative ions for
analysis. Positive ions are made from regular direct
photoionization, but negative ion formation requires a higher
powered laser and a secondary process to gain electrons. Most of
the molecules that come off the sample site are neutrals, and thus
can attract electrons based on their electron affinity. The
negative ion formation process is less efficient than forming just
positive ions. The sample constituents will also affect the outlook
of negative ion spectra.
[0062] Other advantages with the LDLPMS method include the
possibility of constructing the system to give a quiet baseline of
the spectra because one can prevent coevolved neutrals from
entering the flight tube by operating the instrument in a linear
mode. Also, in environmental analysis, the salts in the air and as
deposits will not interfere with the laser desorption and
ionization. This instrumentation also is very sensitive, known to
detect trace levels in natural samples without any prior extraction
preparations.
E. MALDI-TOF-MS
[0063] Since its inception and commercial availability, the
versatility of MALDI-TOF-MS has been demonstrated convincingly by
its extensive use for qualitative analysis. For example,
MALDI-TOF-MS has been employed for the characterization of
synthetic polymers, peptide and protein analysis (Zaluzec et al.,
Protein Expr. Purif., 6:109, 1995; Roepstorff et al., EXS, 88:81,
2000), DNA and oligonucleotide sequencing, and the characterization
of recombinant proteins. Recently, applications of MALDI-TOF-MS
have been extended to include the direct analysis of biological
tissues and single cell organisms with the aim of characterizing
endogenous peptide and protein constituents. Li et al., Trends
Biotechnol., 18:151 (2000); Caprioli et al., Anal. Chem., 69:4751
(1997).
[0064] The properties that make MALDI-TOF-MS a popular qualitative
tool--its ability to analyze molecules across an extensive mass
range, high sensitivity, minimal sample preparation and rapid
analysis times--also make it a potentially useful quantitative
tool. MALDI-TOF-MS also enables non-volatile and thermally labile
molecules to be analyzed with relative ease. It is therefore
prudent to explore the potential of MALDI-TOF-MS for quantitative
analysis in clinical settings, for toxicological screenings, as
well as for environmental analysis. In addition, the application of
MALDI-TOF-MS to the quantification of polypeptides (i.e., peptides
and proteins) is particularly relevant. The ability to quantify
intact proteins in biological tissue and fluids presents a
particular challenge in the expanding area of proteomics and
investigators urgently require methods to accurately measure the
absolute quantity of proteins. While there have been reports of
quantitative MALDI-TOF-MS applications, there are many problems
inherent to the MALDI ionization process that have restricted its
widespread use. Wang et al., J. Agric. Food. Chem., 48:3330 (2000);
Desiderio et al., Biopolymers, 40:257 (1996). These limitations
primarily stem from factors such as the sample/matrix
heterogeneity, which are believed to contribute to the large
variability in observed signal intensities for analytes, the
limited dynamic range due to detector saturation, and difficulties
associated with coupling MALDI-TOF-MS to on-line separation
techniques such as liquid chromatography. Combined, these factors
are thought to compromise the accuracy, precision, and utility with
which quantitative determinations can be made.
[0065] Because of these difficulties, practical examples of
quantitative applications of MALDI-TOF-MS have been limited. Most
of the studies to date have focused on the quantification of low
mass analytes, in particular, alkaloids or active ingredients in
agricultural or food products, whereas other studies have
demonstrated the potential of MALDI-TOF-MS for the quantification
of biologically relevant analytes such as neuropeptides, proteins,
antibiotics, or various metabolites in biological tissue or fluid.
Muddiman et al., Fres. J. Anal. Chem., 354:103 (1996); Nelson et
al., Anal. Chem., 66:1408 (1994). In earlier work it was shown that
linear calibration curves could be generated by MALDI-TOF-MS
provided that an appropriate internal standard was employed. Duncan
et al., Rapid Commun. Mass Spectrom., 7:1090 (1993). This standard
can "correct" for both sample-to-sample and shot-to-shot
variability. Stable isotope labeled internal standards
(isotopomers) give the best result.
[0066] With the marked improvement in resolution available on
modern commercial instruments, primarily because of delayed
extraction (Bahr et al., J. Mass. Spectrom., 32:1111, 1997), the
opportunity to extend quantitative work to other examples is now
possible; not only of low mass analytes, but also biopolymers. Of
particular interest is the prospect of absolute multi-component
quantification in biological samples (e.g., proteomics
applications).
[0067] The properties of the matrix material used in the MALDI
method are important. Only a select group of compounds is useful
for the selective desorption of proteins and polypeptides. A review
of all the matrix materials available for peptides and proteins
shows that there are certain characteristics the compounds must
share to be analytically useful. With a few exceptions, most of the
matrix materials used are solid organic acids. Liquid matrices have
also been investigated, but are not used routinely.
Automation and High-Throughput Analysis
[0068] In some embodiments of the invention, one or more steps of
the method can be automated and implemented for the analysis of a
very large number of samples. For example, the step of depositing
one or more portions of a hydrogel mixture can be done robotically.
Likewise, removing the digested mixture and extracting the
polypeptides can be done robotically. Commercially available mass
spectrometry system (e.g., MALDI instruments) can record the mass
spectra of all the extract samples in quick succession. The
analysis of the data can also be automated by employing a computer
program to analyze generated data. With sufficient automation, a
single person, with access to a MALDI instrument could use the
automated techniques to measure as many as 1000 samples per
day.
[0069] In some embodiments, the one or more hydrogel portions are
placed in a regular column and row pattern (e.g., corresponding to
that found in a standard 96 well plate) in a highly automated
fashion, thereby ensuring that the rate of screening is dependent
only on the speed of sequential analysis of the mass spectrometer.
An automatic sampler can be used to transport samples between the
purification system (which includes extraction and/or column
purification) and the mass spectrometer. Autosamplers can be
purchased from standard laboratory equipment suppliers such as
Gilson and CTC Analytics. Such samplers function at rates of about
10 seconds/sample to about 1 min/sample. In some embodiments, the
invention also includes a computer and software operably coupled to
the apparatus for recording and analyzing mass spectrometer data
and for controlling the automatic sampler.
[0070] In some embodiments, the method of analyzing polypeptide
content is a high-throughput method. "High throughput mass
spectrometry" is used herein to refer to a mass spectrometry system
that is capable of analyzing samples at a rate of from about 100 or
200 samples per day to about 15,000 samples per day. In general,
mass spectrometry and MALDI-MS in particular have proven to be
highly amenable to high throughput applications in both clinical
and basic research settings. For example, Sequenom Inc. (San Diego,
Calif.) has established MALDI-MS as an effective technique in the
field of genotype profiling, and is providing diagnostic products
in this area. In some embodiments, the method is capable of
analyzing about 200 samples in less than an hour, e.g., 200 samples
are injected into a mass spectrometer and analyzed in less than an
hour. High throughput screening preferably takes advantage of the
ability to automate the data acquisition and data analysis
methods.
Applications
[0071] Alterations in proteins abundance, structure, or function,
act as useful indicators of pathological abnormalities prior to
development of clinical symptoms and as such are often useful
diagnostic and prognostic biomarkers. The analysis of polypeptide
content of animal tissue can be used for various different
purposes. Hanash, S., Nature 422, 226-232 (2003). For example, by
examining the proteome of various tissues, one can identify
subjects that have or are at risk of disease, including infections,
cancer, autoimmune disorders, diabetes, or virtually any other
condition for which protein aberrations are known. In many cases,
the underlying mechanisms of diseases such as cancer are quite
complicated in that multiple dysregulated proteins are involved. In
other embodiments, analysis of polypeptide content of animal tissue
can be used in drug development to identify regulated targets and
evaluate drug effects.
[0072] In one aspect, the present invention involves the use of
mass spectroscopy to diagnosis or predict conditions or disease
states in a subject. Ideally, the use of the present invention
permits replicate sampling to ensure accuracy, but also permits
testing for multiple targets in discrete but spatially related
portions of a tissue. Tissue samples may be obtained using
protocols described herein.
[0073] Conditions that may be diagnosed according to the present
invention include, but are not limited to, cancer, infection,
congenital disease, exposure to toxicity, and diabetes. Generally,
the protein expression of one or more protein targets in the tissue
sample will be compared in a standard or known expression level,
array or distribution. Alternatively, known healthy tissue may be
interrogated in parallel to provide the "normal control" to which
the sample is compared.
[0074] In another embodiment, the present invention permits the
monitoring of disease development, disease progression, or the
effects of a treatment on a subject. Such an assay will comprise,
essentially, the same steps a diagnostic method with the exception
that the timing of the examination will be based on (a) a previous
negative diagnostic result, (b) a previous positive diagnostic
result, or (c) a prior treatment application.
[0075] The present invention is illustrated by the following
examples. It is to be understood that the particular examples,
materials, amounts, and procedures are to be interpreted broadly in
accordance with the scope and spirit of the invention as set forth
herein.
EXAMPLES
Example 1
Localized In-Situ Hydrogel-Mediated Protein Digestion and
Extraction Technique for on-Tissue Analysis
[0076] Identification of imaged small molecules (e.g.
pharmaceuticals, peptides and lipids) commonly proceeds with
on-tissue tandem MS (MS/MS) and accurate mass measurements. These
experiments usually follow an initial IMS experiment and thus are
carried out on the section already imaged or on an adjacent serial
tissue specimen. This allows for the best co-localization of the
initial imaged precursor ion to the confident MS/MS identification
from product ions. However, on-tissue protein identification can be
laborious. One approach utilizes traditional proteomic
methodologies to identify proteins. Franck et al., Mol. Cell. Prot.
8:2023-2033 (2009). The first step may involve one of several
approaches: microextraction from the tissue surface with
aqueous/organic solvents, tissue homogenization from multiple
neighboring tissue specimens, laser capture microdissection (LCM)
collection of regions of interest, or bulk tissue homogenization
depending on the localization and predicted relative amount of the
desired protein. High-performance liquid chromatography (HPLC)
fractionation of this bulk tissue homogenization often follows
where the fractions are collected throughout the gradient. Aliquots
of these fractions are spotted onto a MALDI target for MS analysis
to determine what fractions contain the proteins of interest. The
aliquot may be subjected to additional offline clean-up and
separations such as sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) to further isolate the approximate
molecular weight fraction in a gel band. A standard in-gel
proteolytic digestion procedure is performed, and the product
peptides are identified via HPLC-MS/MS and database searching. This
process works well for many soluble and more abundant protein
species and in applications where there is a relatively large
amount of tissue available. However, the process is time consuming,
sample (tissue) quantities are often limited and isolating distinct
micro-regions of tissue for spatially-directed analysis can be
difficult.
[0077] A second approach for on-tissue protein identification is
the use of in-situ proteolytic digestion. Groseclose, J. Mass
Spectrom. 42:254-262 (2007). Typically, serial sections of tissue
are employed. On one tissue specimen, the IMS experiment is
performed targeting proteins, peptides, lipids or other classes of
molecules. On an adjacent serial tissue specimen a proteolytic
enzyme (e.g. trypsin) is deposited in a similar array to that of
the imaged section. After the enzyme is applied and digestion is
allowed to proceed, application of a MALDI matrix follows for
peptide imaging. The protein and peptide images are correlated
using post-processing tools to direct subsequent on-tissue MS/MS
analyses. This approach correlates the original ion image with
protein identification through peptide analysis at identical
locations on the sections. A drawback of this approach includes the
poor on-tissue digestion efficiency caused from rapid drying of the
small droplets of enzyme solution that are required to achieve high
spatial resolution. Increasing the aqueous content of the enzyme
solution and/or spraying/depositing larger droplets can mitigate
the problem, but this may lead to delocalization of endogenous
biological molecules which will make image correlation and
confident identification challenging. Additionally, the total
number of tryptic peptides relating to the desired protein species
may be limited, reducing the confidence of identification.
Previously reported methods for in-situ digestion on tissue
specimens do not typically probe as deeply into the proteome when
compared to bulk homogenates analyzed by HPLC-MS/MS identification.
Yao et al., Proteom. 8:3692-3701 (2008).
[0078] The present example describes a spatially-directed
simultaneous on-tissue proteolytic digestion and extraction
technique to be used in conjunction with existing MALDI IMS
workflows. See Harris et al., Anal. Chem. 85, 2717-23 (2013). This
approach utilizes on-tissue protein identification within a
hydrogel microreactor network to simultaneously digest and extract
proteins and peptides followed by traditional peptide sequence
analysis.
EXPERIMENTAL
Reagents
[0079] For hydrogel synthesis, alginic acid sodium salt was
purchased from Alfa Aesar (Ward Hill, Mass. USA) and calcium
chloride dihydrate was purchased from J. T. Baker (Center Valley,
Pa. USA). The hydrogel additives Triton X-100, ammonium bicarbonate
and proteomics grade trypsin from porcine pancreas (dimethylated),
the MALDI matrices 2,5-dihydroxybenzoic acid (DHB, 98%) and
sinapinic acid (98%) and the acids trifluoroacetic and formic acid
and were all purchased from Sigma Aldrich (St. Louis, Mo. USA).
Solvents (ethanol, xylenes, methanol and acetonitrile) were all
HPLC grade and the histological dyes (hematoxylin and eosin) were
purchased from Fisher Scientific (Fairlawn, N.J. USA). 18 M.OMEGA.
water was provided via a Millipore Milli-Q Synthesis A10
(Billerica, Mass. USA). All reagent listed were used without
additional purification.
Hydrogel Fabrication
[0080] Hydrogels were fabricated using a previously described
method utilizing template chromatography paper (Whatman,
Buckinghamshire, UK). Bracher et al., ACS App. Mat. Inter.
1:1807-1812 (2009); Bracher et al., Adv. Mat. 21:445-450 (2009);
Bracher et al., Soft Mat. 6:4303-4309 (2010). Briefly, designs of
various shapes and sizes were created in computer software and
color laser printed (Bizhub C360, Konica Minolta, Ramsey, N.J. USA)
onto the chromatography paper (20.times.20 cm). The color levels
were increased to maximize the amount of ink printed. The paper was
reprinted with an identical pattern for a total of 3 times to
ensure a thick ink coating. The paper template was then cut out and
heated for 120 sec on each side with a heat gun (low setting
.about.300.degree. C., 1400 W Milwaukee heat gun, Brookfield, Wis.
USA). This allowed for the hydrophobic color ink to melt and
penetrate into the paper for defined hydrophobic boundaries. The
patterned templates were then soaked in 500 mM CaCl.sub.2 for 60
sec as the free Ca.sup.2+ ions act as a crosslinking agent to
penetrate into the applied polymer creating an ionotropic hydrogel.
The hydrogel polymer (alginate) was diluted to 2% w/v in water. The
polymer solution was mixed with 200 mM ammonium bicarbonate with
0.02% Triton X-100 in a 1:1 ratio such that the final polymer
solution was 1% w/v in 100 mM ammonium bicarbonate and 0.01% Triton
X-100. This solution was first mixed with trypsin prior to being
spotted onto the calcium soaked pattern templates. The volume
deposited (V.sub.tot) and the amounts of trypsin in each digestion
are outlined per experiment later herein.
Tissue Specimening and Pre-Treatment
[0081] Brains from Sprague-Dawley rats were collected and stored at
-80.degree. C. prior to sectioning (Pel-Freez Biologicals, Rogers,
Ark. USA). Coronal and sagittal sections (12 and 9 .mu.m,
respectively) were taken at -19.degree. C. on a cryostat (Leica
Microsystems Inc., Bannockburn, Ill. USA). Tissue specimens were
thaw mounted onto premium microscope slides (Fisher Scientific,
Fairlawn, N.J. USA) which were cleaned previously in 100% ethanol
(twice) and 100% methanol rinses (60 sec each).
Protein Digestion, Extraction and Identification
[0082] The ionotropic hydrogel mixture was spotted onto the paper
target where it was polymerized (<5 minutes) (FIG. 1a). The gel
was then removed from the paper pattern via a spatula, thin nosed
tweezers or suction via a pipette tip and placed onto a mounted
piece of tissue (FIG. 1b). To retain the enzyme activity and
improve digest efficiency, the slide was placed in a glass petri
dish with a small (4 cm.times.4 cm) wetted (250-1000 .mu.L
depending on gel and tissue sizes to minimize condensation on
tissue) piece of paper towel underneath the slide. The petri dish
was closed with the glass lid and wrapped with parafilm to make an
air-tight seal. The petri dish was placed in an oven and heated to
a desired temperature (e.g. 50.degree. C.) for 6-12 hours depending
on the tissue thickness, size/shape of the gel, enzyme, etc. This
process ensured that the hydrogel did not become dry during
digestion. After the digestion, the gel was removed and placed in a
micro-centrifuge tube to undergo organic (50% acetonitrile/5%
formic acid) and aqueous (50 mM NH.sub.4HCO.sub.3, 25 mM
CaCl.sub.2) solvent extractions (process repeated twice) to shrink
and swell the hydrogel to extract digested peptides (FIG. 1c). The
supernatants of each extraction are combined and dried in a
centrifugal vacuum concentrator (SPD Speedvac, Thermo Scientific,
Waltham, Mass. USA). The reconstituted extract (50-75 .mu.L in 0.1%
formic acid) was then spotted for MALDI MS (FIG. 1d) and LC-MS/MS
(FIG. 1e) and could be stored at -80.degree. C. All microscopy was
performed with an Olympus BX 50 (Center Valley, Pa. USA) with the
following histology procedure: 95% ethanol 30 sec, purified water
30 sec, hematoxylin 120 sec, water 15 sec, 70% ethanol 15 sec, 95%
ethanol 15 sec, eosin 60 sec, 95% ethanol 15 sec, 100% ethanol 15
sec, xylenes 120 sec.
Mass Spectrometry Analysis
[0083] MALDI MS profiling was conducted by spotting 2 .mu.L of a
1:1 mix of reconstituted digest mix with 20 mg mL.sup.-1 sinapinic
acid in 90% acetonitrile and 0.25% trifluoroacetic acid. A Bruker
Ultraflextreme.TM. mass spectrometer (Bremen, Germany) was used to
acquire spectra with the spot size set to medium, laser energy at
78% and laser frequency set to 500 Hz. Resulting spectra were an
average of multiple laser shots. This data was processed using
FlexAnalysis v3.3.
[0084] MALDI IMS experiments were performed with a Waters
Synapt.TM. G2 instrument (Manchester, UK) at laser frequency 1000
Hz, 1 scan sec.sup.-1 and 125 .mu.m.times.125 .mu.m laser step. MS
acquisition was in Resolution mode with trap and transfer collision
energies set to 4 V and 2 V, respectively. Traveling wave ion
mobility settings were as follows: nitrogen gas flow at 65 mL
min.sup.-1 (2.5 mbar), helium cell gas flow at 165 mL min.sup.-1,
450 .mu.s pulse delay, ion mobility wave height 40 V, variable ion
mobility wave velocity starting at 1100 m s.sup.-1 and ending 400 m
s.sup.-1 with the velocity ramped over 100% of the cycle. DHB was
applied with an automated sprayer at a concentration of 5 mg
mL.sup.-1 in 90% acetonitrile and 0.25% trifluoroacetic acid for a
total of 4 passes (HTX Technologies TM Sprayer, Carrboro, N.C.
USA). On-tissue MS/MS was performed at designated collision
energies (32.5 V for PC 36:1, 35 V for PC 34:1 and 37.5 V for PC
36:4) applied in the trap triwave after precursor ion selection in
the quadrupole. Data was processed in Waters HD Imaging software
(images normalized to total ion current), Driftscope v2.2 and
Masslynx.
[0085] Extracted peptides were analyzed by a 75 min data dependent
LC-MS/MS analysis. Briefly, peptides were loaded via pressure cell
onto a 40 mm by 0.1 mm self-packed reversed phase (Jupiter 5 .mu.M,
300 .ANG. C.sub.18--Phenomenex, Torrance, Calif. USA) trapping
column fritted into an M520 filter union (IDEX, Lake Forest, Ill.
USA). After loading and equilibration, this trapping column was
attached to a 200 mm by 0.1 mm (Jupiter 3 .mu.m, 300 .ANG.
C.sub.18--Phenomenex, Torrance, Calif. USA), self-packed analytical
column with a laser pulled tip (i.d. 1 .mu.m) coupled directly to
an LTQ linear ion trap (Thermo Scientific, Waltham, Mass. USA)
using a nanoelectrospray source. Reverse phase separation was
performed on a Waters nanoAcquity.TM. UPLC (Milford, Mass. USA)
using the following gradient run at 500 nL min.sup.-1 flow rate:
initial flow of 98% A (0.1% formic acid), 2% B (acetonitrile, 0.1%
formic acid) ramped to 25% B over 45 min, to 90% B at 60 min where
it remained for 5 min, and then a 2 min ramp back to 2% B where it
remained for an additional 8 min. A series of full scan mass
spectra followed by 5 data-dependent MS/MS spectra was collected
throughout the run and dynamic exclusion was enabled to minimize
acquisition of redundant spectra. MS/MS spectra were searched via
SEQUEST against a rat database (UniprotKB taxon 10116--reference
proteome set) that also contained reversed versions for each of the
entries. Yates et al., Anal. Chem. 67:1426-1436 (1995).
Identifications were filtered and collated at the protein level
using Scaffold (Proteome Software, Portland, Oreg. USA) with a 0.5%
false discovery recovery rate.
Results and Discussion
[0086] A workflow for a typical hydrogel mediated on-tissue
digestion is shown in FIG. 1. A circular 4 mm hydrogel containing
trypsin (V.sub.tot=18 .mu.L, 20 .mu.g trypsin in 100 mM
NH.sub.4HCO.sub.3) was placed on a 12 .mu.m thick section of rat
cerebrum and allowed to react for 6 hours in a humidity chamber at
50.degree. C. Following digestion, the gel underwent solvent
extraction for peptide recovery, and the resulting peptides were
dried down and reconstituted to a volume of 75 L. MALDI MS spectra
(2000 shots summed, linear mode) of a blank (trypsin loaded
off-tissue, FIG. 2a), control (no trypsin on-tissue, FIG. 2b) and
digested (trypsin loaded on-tissue, FIG. 2c) hydrogels obtained
from 2% (by volume) of the total extract. Abundant signals were
present throughout the mass range demonstrating extensive on-tissue
digestion and efficient extraction from the hydrogel network of the
trypsin loaded on-tissue hydrogel (FIG. 2c). The absence of signals
from the hydrogel polymer is the result of the presence of
CaCl.sub.2 in the extraction buffer which maintains the integrity
of the ionotropic hydrogel, and prevents contamination of the
supernatant. For a blank, a hydrogel with trypsin was placed on an
empty microscope slide (FIG. 2a) and as a control, a gel without
trypsin was placed on a serial section of tissue (FIG. 2b). As
shown in the blank spectrum (FIG. 2a), no signal is present
indicating autolytic trypsin peptides are below the detection limit
for the amount of trypsin used in this study. The control spectrum
shows a lack of peptide signals in the lower m/z 1500-4000 range,
but some low intensity signals in the higher mass range (m/z
4500-9000) were observed, e.g. m/z 4964 and 8566 (FIG. 2b inserts).
It is likely that these signals are from the abundant and commonly
observed intact proteins thymosin .beta.4 and ubiquitin,
respectively. Rahman et al., Can. Res. 71:3009-3017 (2011).
Interestingly, the presence of these ions indicates that abundant
smaller proteins can passively diffuse into these gels as well.
Potential modifications of the procedure to improve the extraction
of intact proteins into the gels include increasing the porosity
(Wu et al., Chem. Rev. 112:3959-4015 (2012)) or altering the
alginate (or other hydrogel) properties. Pawar S N, Edgar K J.,
Biomat, 33:3279-3305 (2012). This could be of particular usefulness
in off-line solution digestions of hydrogel extracted proteins and
top-down protein identification workflows.
[0087] The LC-MS/MS analysis of this sample digestion identified
211 proteins. This amount is in range with similarly sized punched
sections of mouse liver fresh frozen and formalin-fixed and
paraffin-embedded tissues analyzed by LC MALDI FTICR and TOF/TOF
analysis (Aemi et al., Anal. Chem. 81:7490-7495 (2009)), and
comparable to simple protein extractions without detergent
additives from bulk homogenization of mouse brains analyzed by
LC-MS/MS. Shevchenko et al., J. Prot. Res. 11:2441-2451 (2012).
There were 85 proteins with extensive sequence coverage, having
more than 5 unique peptides identified. Also of interest were high
molecular weight proteins that are not readily observed in typical
MALDI IMS experiments (Table 1). For example, 26 large proteins
were confidently identified with predicted molecular weights
greater than 100 kDa. Many of these higher molecular weight
proteins were identified as membrane proteins (e.g. several Na+/K+
transporting ATPases, neurofascin, clathrin heavy chain and
ankyrin-2), cytoskeletal proteins (e.g. a actinin,
microtubule-associated proteins and spectrin), extracellular matrix
proteins (restrictin) and cytoplasmic proteins (e.g.
puromycin-sensitive aminopeptidase, protein bassoon and dynein
heavy chain) Another 77 of the 211 proteins were detected in the
50-99 kDa range, also a challenging intermediate high mass range
for MALDI IMS.
TABLE-US-00001 TABLE 1 High molecular weight (>100 kDa) proteins
identified with on-tissue hydrogel-mediated protein digestion and
extraction on rat brain cerebrum. UniProt Molecular # # Accession
Weight Unique Assigned Protein Name Number (kDa) Peptides Spectra
Hexokinase type 1 P05708 102 4 6 .alpha. actinin 1 Q6GMN8 103 5 10
Puromycin-sensitive P55786* 103 4 6 aminopeptidase Type II brain
4.1 minor Q9JMB2 107 3 5 isoform Na.sup.+/K.sup.+ ATPase .alpha.3
subunit P06687 112 20 67 Na.sup.+/K.sup.+ transporting D3ZSA3 112 3
6 ATPase .alpha.2 chain precursor Na.sup.+/K.sup.+ transporting
P06685 113 10 17 ATPase .alpha.1 chain precursor Contactin-1
precursor Q63198 113 6 9 Neurofilament heavy F1LRZ7 114 8 11
polypeptide Ubiquitin-activating Q5U300 118 3 4 enzyme E1
Electroneutral K.sup.+/Cl.sup.- Q63633 124 2 2 cotransporter 2
Plasma membrane P11506 137 6 9 Ca.sub.2.sup.+ ATPase isoform 2
Neurofascin P97685 138 2 2 Plasma membrane Ca.sub.2.sup.+ P11505
139 3 4 transporting ATPase 1 Ras GTPase-activating Q9QUH6 143 3 3
protein SynGAP Tenascin-R precursor Q92752* 150 2 3 (Restrictin)
Clathrin heavy chain 1 P11442 191 9 13 Microtubule-associated
P15146 199 21 46 protein 2 Microtubule-associated P15205 270 8 13
protein 1B Spectrin .beta. chain, brain 2 Q9QWN8 271 5 7
Non-erythroid spectrin .beta. Q6XD99 274 29 47 Spectrin .alpha.
chain, brain P16086 285 60 109 Microtubule-associated G3V7U2 300 15
27 protein 1A Protein bassoon O88778 418 2 4 Ankyrin-2 Q01484* 437
5 5 Dynein heavy chain, P38650 532 8 9 cytosolic *Predicted protein
based on sequence homology from Homo sapiens
[0088] The hydrogels can also be used for a sequential analysis
workflow in conjunction with a previously imaged section of tissue.
In this case, the digestion and extraction occur after the initial
IMS and on-tissue MS/MS, but before histological staining (FIG. 3).
A 9 .mu.m thick section of rat brain was imaged with the traveling
wave ion mobility spectrometry (TWIMS) separation. The use of TWIMS
in MALDI IMS experiments separates MALDI matrix ions from the
targeted biological ions. McLean et al., J. Mass Spectrom.
42:1099-1105 (2007); Djidja et al., J. Prot. Res. 8:4876-4884
(2009). Shown in FIG. 3a is an overlaid ion image of three
identified lipid species assigned to phosphatidylcholines (PC) at
m/z 788.62, 798.54 and 804.55, relating to PC 36:1, PC 34:1 and PC
36:4, respectively. These species were identified based on accurate
mass and MS/MS (FIG. 3b-d) of neighboring pixels after the initial
full scan IMS experiment, and confirmation that these ions fell
into the lipid trend line in the ion mobility dimension of
separation.
[0089] After the lipid IMS and MS/MS experiments were completed,
the tissue was not visibly damaged since the laser energy used
(energy level 275, approximately 46% power) was sufficiently high
enough to obtain signal, but below the threshold for ablation of
the tissue. Before proceeding to the in-situ digestion, the tissue
was washed two times for 30 sec (70% ethanol and 100% ethanol,
respectively) to remove the residual DHB matrix, salts and lipids
on the tissue surface. A hydrogel disc (diameter=1.5 mm,
V.sub.tot=8 .mu.L containing 10 .mu.g trypsin and 0.1% Triton X-100
in 100 mM NH.sub.4HCO.sub.3) was placed on the cerebellum region of
the tissue and incubated for 6 hours at 50.degree. C. For
comparison, another imaged rat brain tissue specimen was washed and
a 1.5 mm (diameter) region of cerebellum was removed for an
in-solution digestion (V.sub.sol=250 .mu.L) with the same amount of
trypsin and Triton X-100. A third imaged tissue specimen was
treated the same as the previous two, but a hydrogel without
trypsin was placed on the cerebellum as a control. After 6 hours of
incubation, the control gel was placed in a 250 .mu.L solution with
10 .mu.g trypsin and 0.1% Triton X-100 in 100 mM NH.sub.4HCO.sub.3
for 6 hours at 50.degree. C. All samples were dried and
reconstituted to identical volumes (V=50 .mu.L), and the samples
were analyzed via LC-MS/MS.
[0090] The on-tissue digestion using the hydrogel protocol
identified 96 proteins compared to 147 for the in-solution tissue
digestion and 22 proteins for the control hydrogel solution
digestion. The identified high molecular weight (>100 kDa)
proteins again reveals many membrane (e.g. hexokinase-1, several
Na+/K+ transporting ATPases, neural cell adhesion molecule 1, etc.)
and cytoskeletal proteins (e.g. microtubule-associated proteins,
neurofilament heavy polypeptide, spectrin, etc.) identified using
the homogenization and hydrogel digestions (Table 2). However, the
digestion efficiencies of the two approaches differed. Based on the
number of unique peptides identified and the number of assigned
spectra, the in-solution homogenization digestion was a more
extensive, albeit destructive, digestion procedure compared to the
current iteration of the hydrogel digestion. However, given that
the same amount of trypsin was used in both methods, the difference
may be attributed to the limited volume of the hydrogel used
on-tissue (8 .mu.L) that was more than 30.times.smaller compared to
the solution volume of the homogenization (250 .mu.L), and the
requirement for proteins and peptides to migrate into the hydrogel
during the digestion incubation. The accessibility of proteins for
digestion was greater in the homogenization procedure since trypsin
mobility in solution is greater than in the hydrogel network, and
peptides are released into solution rather than required to diffuse
from tissue into the hydrogels. Future iterations of on-tissue
hydrogels can be produced to reduce the polymer network density by
using alternative hydrogel compositions and creating gels with
alternative geometries to increase the total volume-to-surface area
ratio of the structure.
TABLE-US-00002 TABLE 2 Comparison of the high molecular weight
(>100 kDa) proteins identified with an in-solution
homogenization protocol and an on-tissue hydrogel-mediated protein
digestion on a previously imaged rat brain cerebellum. In-solution
On-tissue Homogenization Hydrogel UniProt Molecular # # # # Protein
Accession Weight Unique Assigned Unique Assigned Name Number (kDa)
Peptides Spectra Peptides Spectra Microtubule-associated Q63560 100
6 6 4 4 protein 6 Hexokinase-1 P05708 102 3 3 3 4 Na.sup.+/K.sup.+
transporting P06687 112 18 24 8 9 ATPase subunit .alpha. 3
Na.sup.+/K.sup.+ transporting P06686 112 7 8 2 2 ATPase subunit
.alpha. 2 Na.sup.+/K.sup.+ transporting P06685 113 6 7 2 2 ATPase
subunit .alpha. 1 Contactin-1 Q63198 113 3 3 3 4 Neurofilament
heavy F1LRZ7 114 7 9 2 2 polypeptide Sarcoplasmic/endoplasmic
P11507 115 4 4 2 2 reticulum Ca.sup.2+ ATPase 2 Neural cell
adhesion F1LUV9 119 5 6 3 3 molecule 1 Plasma membrane Ca.sup.2+
P11506 137 7 9 7 7 transporting ATPase 2 Clathrin heavy chain 1
D4AD25 192 14 16 2 2 Microtubule-associated P15146 202 6 6 2 2
protein 2 Microtubule-associated F1LRL9 270 5 5 3 3 protein 1 light
chain LC1 Spectrin .beta. 2, isoform G3V6S0 274 20 20 9 10 CRA_a
Spectrin .alpha. chain, brain E9PU22 285 38 41 10 11
Microtubule-associated P34926 300 4 5 2 2 protein 1A Cytoplasmic
dynein 1 P38650 532 6 6 3 3 heavy chain 1
[0091] The hydrogel digested tissue specimens can be used for
additional analysis such as histological staining (hematoxylin and
eosin stain, H & E) for evaluation of cells and surrounding
tissue regions. The hydrogel-digested tissue was washed, stained
and analyzed by microscopy (FIG. 4). A serial rat brain section
that had not been imaged or treated previously was stained with H
& E by the identical procedure for comparison purposes (FIG.
4a). It is apparent that there are differences between the two
stained tissue specimens mainly in the cellular density. There are
a lower number of nuclei (stained blue/purple by hematoxylin)
present in the imaged and extracted tissue specimen compared to an
untreated section (FIG. 4b). The losses are due to a combination of
cellular material being extracted into the hydrogel during
incubation/digestion, and the extensive washing steps prior to
staining which may have dislodged cellular material from the slide
surface. The loss of tissue into the hydrogel is a critical insight
into the current function of the gels. Future refinements to these
materials will focus on enhancing extraction of already imaged
cells and cellular material to maximize proteomic coverage and/or
minimizing the observed tissue damage while still obtaining useful
proteomic information to be used in conjunction with histological
examination of the same tissue specimen. Nonetheless, differential
identification of regions (e.g. extent of myelination, white and
grey matter regions, location of granular cells) within the tissue
was possible in this first study using the protocol for H & E
staining. The inventors are developing new staining protocols that
are amenable to the pretreatment of the tissue specimens to prevent
any potential damage to the tissue specimen.
CONCLUSION
[0092] Hydrogel mediated on-tissue digestion has been demonstrated
to be a simple and inexpensive tool to be added to existing IMS
methods for protein identification. The hydrogels used in this
study are rapidly formed, and can be shaped to match the size of
the particular tissue region in question. This example demonstrates
that sequential experiments can be performed on a single tissue
specimen, i.e. IMS, on-tissue MS/MS, protein identification via
LC-MS/MS and histology.
Example 2
Histology-Directed Microwave Assisted Enzymatic Protein Digestion
for MALDI MS Analysis of Mammalian Tissue
[0093] This example presents an on-tissue proteolytic digestion and
peptide extraction method using microwave irradiation for in situ
analysis of proteins from spatially defined regions of a tissue
specimen. The methodology utilizes hydrogel discs (1 mm diameter)
imbibed with trypsin solution. The hydrogel discs are applied to a
tissue specimen, directing enzymatic digestion to a spatially
confined area of the tissue. By incorporating applying microwave
radiation, protein digestion takes place is performed on-tissue in
2 minutes on-tissue, and the extracted peptides are then analyzed
via MALDI MS and LC-MS/MS. The reliability and reproducibility of
the microwave-assisted hydrogel mediated on-tissue digestion was
demonstrated by the comparison with other on-tissue digestion
strategies, including comparisons with conventional heating and
in-solution digestion. All the experiments were replicated and
LC-MS/MS data were evaluated considering the number of identified
proteins as well as the number of protein groups and of distinct
peptides. The results of this study demonstrate that a rapid and
reliable protein digestion can be performed on a single thin tissue
specimen while preserving the tissue architecture, and the
resulting peptides are extracted in sufficient abundance to permit
robust analysis using LC-MS/MS. An overview of the workflow
involved is shown in FIG. 5. This approach will be most useful for
samples that have limited availability but are needed for multiple
analyses, especially for the correlation of proteomics results data
with histology and immunohistochemistry.
Materials and Methods
[0094] For the hydrogel synthesis acrylamide/bisacrylamide was
purchased from Biorad Life Sciences (Hercules, Calif., USA), while
ammonium persulfate was purchased from Sigma Aldrich (St. Louis,
Mo., USA). The hydrogel additives, ammonium bicarbonate, the MALDI
matrix alpha-cyano-4-hydroxycinnamic acid (98%), the acids
trifluoroacetic (TFA) and formic (FA) were all purchased from Sigma
Aldrich (St. Louis, Mo., USA). Punch biopsies were purchased from
Acuderm, Inc. (Ft. Lauderdale, Fla.). Mass spectrometry grade
Trypsin Gold was purchased from Promega Corporation (Madison, Wis.,
USA). HPLC grade solvents (ethanol, xylenes, methanol, and
acetonitrile) and histological dyes (hematoxylin and eosin) were
purchased from Fisher Scientific (Fairlawn, N.J., USA). Xylene was
purchased from Acros (Morris Plains, N.J.). Water was provided via
Millipore Milli-Q Synthesis A10 (Billerica, Mass., USA). All
reagents listed were used without additional purification.
Hydrogel Discs Fabrication
[0095] Fabrication of 7.5% polyacrylamide hydrogels was carried out
following and modifying a previously developed procedure: A volume
of 1.24 mL of 30% acrylamide/bisacrylamide solution was added to
1.26 mL TRIS buffer at pH 6.8 and 2.45 mL of water. Nicklay et al.,
Anal Chem. 85(15):7191-6 (2013). The solution was degassed under
vacuum for a minimum of 30 min before adding 50 .mu.L of 10%
ammonium persulfate and of 10 .mu.L of TEMED. The solution was
mixed by inversion and placed into a small Petri dish to polymerize
for 30 min. Finally, punch biopsy tools were used to cut the
microwells in a variety of sizes (1, 1.5, 3 mm diameter). Each
individual microwell of hydrogel was placed in an eppendorf tube,
dried fully in a speedvac and stored at -80.degree. C. until
use.
Tissue Specimening and Pretreatment
[0096] Fresh frozen rat brain was purchased from Pel-Freez
Biologicals (Rogers, Ariz.) and tissue specimens were prepared at 8
.mu.m thickness using a Leica CM3050 cryostat (Leica Microsystems
GmbH, Wetzlar, Germany). Frozen tissue specimens were thaw mounted
on microscope slides or placed into eppendorf tubes (for the
homogenization procedure) and stored in a desiccator until needed.
Each tissue specimen was rinsed using ethanol (95%, 30 sec; 70%, 30
sec) to remove salts, lipids and to obtain optimal sensitivity for
MS analysis of the digested extracts. Seeley et al., J. Am. Soc.
Mass Spectrom. 19, 1069-77 (2008).
On-Tissue Microwave Digestion
[0097] Hydrogels were re-hydrated for 15 mins using 20 .mu.L of 1
.mu.g/mL trypsin (in 100 mM ammonium bicarbonate) and then placed
over the tissue region of interest (brain thalamic region) guided
by the histological features on corresponding serial H&E
stained tissue specimen. The tissue specimens were incubated in a
microwave oven (1.65 kW) for 2 min set at 10% of the power, to
accelerate protein digestion. Each hydrogel disc was removed from
the tissue specimen and placed in separate eppendorf tubes.
Peptides imbibed into the microwell hydrogels were extracted by
organic (50% acetonitrile/5% formic acid) and aqueous (100 mM
ammonium bicarbonate) solvents, a process that was repeated three
times. The supernatant collected from each extraction were combined
and dried in a centrifugal vacuum concentrator (SPD Speedvac,
Thermo Scientific, Waltham, Mass., USA). The reconstituted extracts
(20 .mu.L, 0.1% formic acid) were spotted for MALDI MS analysis and
then stored at -20.degree. C. until LC-MS/MS analysis was
performed.
Other on-Tissue Digestion Strategies
[0098] Further protein digestion experiments were carried out on
serially prepared tissue specimens. First, tissue specimens were
incubated using a conventional oven at 50.degree. C. for 4 hours:
hydrogel discs were still used to allow the digestion to take place
on the brain thalamic region. After digestion, peptides were
extracted from the gel following the same procedure already
described for the microwave digestion. Second, since hydrogels were
fabricated at 1 mm diameter, the rat brain biopsy was also punched
into the thalamic region using a 1 mm punch biopsy tool. This
approach precisely controls the amount of tissue exposed to the
hydrogel and allows for the homogenization and digestion of the
same amount of tissue using conventional sample preparation
methods. Serial sections from the 1 min tissue core were
cryosectioned following the same protocol described above. One set
of digestion experiments was carried out: n=3 tissue core sections
were mounted on microscope slides and hydrogel discs (trypsin
embedded) were placed on top and incubated via conventional oven at
50.degree. C. for 4 hours. Second, n=3 tissue core sections were
marked using a hydrophobic pen and trypsin was manually spotted and
incubated overnight at 37.degree. C. Finally, other serial tissue
core sections from the same tissue specimen were placed into
separate eppendorf tubes and the homogenized as previously
described. Bodzon-Kulakowska et al., J. Chromatogr. B. Analyt.
Technol. Biomed. Life Sci. 849, 1-31 (2007). The digestion was
conducted at 37.degree. C. overnight, and the digested peptides
were extracted following the same procedure described above.
Mass Spectrometry Analysis and Data Processing
[0099] MALDI MS analyses were carried out using an
UltrafleXtreme.TM. MALDI TOF/TOF spectrometer (Broker Daltonics,
Billerica, Mass.) equipped with a SmartBeam.TM. II laser and
operating in positive polarity, reflectron mode. Spectra were
acquired in the range of m/z 500-4000. Flex Control 3.3 software
was used for spectra acquisition. The reproducibility of the
hydrogel-based digestion was evaluated using a set of three
technical replicates within the same tissue sample at different but
histologically identical locations. All spectra were processed
using the same preprocessing procedure to ensure overall
consistency. Briefly, they were baseline-corrected and normalized
according to their total ion current, excluding the top 5% of
intensity values to avoid bias by highly abundant species. The
Mann-Whitney U test and Kruskal-Wallis test were applied to
evaluate statistically significant of differences (protein groups
and distinct peptides) between groups (all different digestion
strategies). The Mann-Whitney U test and Kruskal-Wallis test are
the analogous nonparametric methods of t-test and one-way
between-groups of variance (analysis of variance, ANOVA),
respectively.
LC-MS/MS Analysis
[0100] Resulting peptides were analyzed by a 70 minute data
dependent LC-MS/MS analysis. Briefly, peptides were loaded via
pressure cell onto a 40 mm by 0.1 mm self-packed reversed phase
(Jupiter 5 um, 300 A--Phenomonex) trapping column fritted into an
M520 filter union (IDEX). After loading and equilibration, this
trapping column was attached to a 200 mm by 0.1 mm (Jupiter 3
micron, 300 A), self-packed analytical column coupled directly to
an LTQ (ThermoFisher) using a nanoelectrospray source. A series of
a full scan mass spectrum followed by 5 data-dependent tandem mass
spectra (MS/MS) was collected throughout the run, and dynamic
exclusion was enabled to minimize acquisition of redundant spectra.
MS/MS spectra were searched via SEQUEST against a human database
(UniprotKB--reference proteome set) that also contained a reversed
version for each of the entries. Yates et al., Anal. Chem. 67,
1426-36 (1995). Identifications were filtered to 2 peptides per
protein and 0% peptide false detection rate and collated at the
protein level using Scaffold (Proteome Software). Furthermore,
IDPicker 3 software used to filter the resulting identifications to
a 5% FDR at the peptide level and collate the individual proteins,
requiring a minimum of 2 peptides per protein. Holman et al., Curr
Protoc Bioinforma. 13, unit 13.17 (2012)
Results and Discussion
[0101] The aims of this study were to demonstrate both the
reliability and the relative advantages of the use of microwave
radiation to speed up the on-tissue proteomics workflow and to
demonstrate the methodology to perform the enzymatic digestion in a
histologically defined region on a thin tissue specimen (8 .mu.m).
Various on-tissue digestion strategies were carried out on the same
sample in this study to provide a basis for comparison; therefore,
a series of experiments were designed and carried out using rat
brain serial sections from the same tissue specimen, to avoid
tissue proteome variability. FIG. 6 shows a graphical depiction of
the experimental design used in this study. Fresh frozen rat brain
biopsies were first sectioned at 8 .mu.m thickness and then stained
by hematoxylin and eosin (H&E) for histological evaluation of
regions of interest; the thalamic region was chosen for the
histology-directed experiments. Hydrogel discs, prepared on a prior
day and stored, were reconstituted in the trypsin solution for 15
minutes and then placed on the rat brain thalamic region as
described above. First, two on-tissue digestion experiments were
carried out: FIG. 6a shows two different digestion strategies
(microwave and conventional heating) performed on the whole tissue
specimen using the hydrogel disc for the histology-directed
digestion. FIG. 6b displays another set of experiments performed on
the rat brain thalamic region punched from the bulk biopsy specimen
prior cryosectioning. The bulk specimen was cryosectioned and
stained to provide a visual comparison of the areas sampled for
analysis. The biopsied tissue from the bulk specimen was
cryosectioned at a thickness of 8 .mu.m to collect a precisely
comparable amount of tissue for comparison with the on-tissue
digestion.
[0102] Solvent extracted digested peptides obtained from the first
two hydrogel experiments, carried out on-tissue via microwave and
oven, were reconstituted and mixed with a solution of CHCA for
MALDI MS analyses. The resulting profile MALDI spectra are
displayed in FIG. 7. The peptide spectra are qualitative comparable
with most major ions present in both preparations in the mass range
measured (500-4000 Da). While most peaks are present, there are
notable relative intensity differences, likely due to the different
incubation strategy (microwave vs. conventional oven). This result
should have little impact on downstream identification by LC-MS/MS;
however, these differences would not permit quantitative
comparisons between samples that have been prepared using two
different on-tissue digestion approaches. MALDI MS profiles of
three technically replicated microwave assisted hydrogel mediated
on-tissue digested extracts are presented in FIG. 8. Most of the
signals are detected in all the three replicates, confirming the
reliability of the microwave procedure for the digestion.
[0103] To further validate the on-tissue hydrogel/microwave
digestion approach, other on-tissue digestion experiments were
carried out as described in FIG. 6b. Serial sections at 8 .mu.m
thickness and 1 mm diameter were cut from the thalamic region of
rat brain as described in the experimental section. This experiment
was performed to further validate the extraction localized proteins
by the hydrogel discs through the exposure of a tissue surface cut
at the same diameter which the hydrogel discs were fabricated (1
mm). Rat brain thalamus proteins were digested following three
approaches: 1) using a hydrogel disc and incubating the reaction
for 4 hours at 50.degree. C.; 2) by deposition of the trypsin
solution onto the tissue surface and 3) by the homogenization of
tissue specimens followed by protein extraction; both the last two
approaches were allowed for digestion by overnight incubation at
37.degree. C. (FIG. 6b). All the digested extracts, were analyzed
by LC-MS/MS followed by database search for protein identification.
Data were processed using a 5% FDR, filtering the identified
proteins with number of unique peptides .gtoreq.2 and a p-value
<0.05. Data are summarized in FIG. 9 using Venn diagrams.
[0104] The digestion strategies were evaluated and compared in
different ways: the first comparison was carried out considering
the number of identified proteins found when using microwave
heating compared to conventional heating in an oven. A large number
of proteins (728) were identified in both the microwave digestion
strategy and the oven incubation, both using the hydrogel disc
(FIG. 9a); this finding suggests that the hydrogel disc device
allows for a comparable degree of digestion in 2 minutes using
microwave heating as well as in 4 hours using conventional heating.
Relatively few proteins were uniquely identified in both
approaches, respectively nine from the microwave digestion and ten
from the oven digestion. This finding confirms that by changing the
method of heating to microwave irradiation, the protein population
sampled from the brain tissue was not altered. Moreover, FIG. 9b
displays a Venn diagram comparing the number of identified proteins
within the experiments performed using the 1 mm diameter tissue
specimens from the thalamic region of the rat brain. Also in this
case, the majority of the identified proteins (695) were identified
using both digested extracts (from the hydrogel disc and from the
tissue homogenization), while a few proteins were found uniquely
expressed into the two set of samples, respectively five from the
sample from the hydrogel digestion and eight from the
homogenization process (FIG. 9b). Since homogenization is
considered to be the most comprehensive method of protein
extraction from tissue, allowing for the most complete disruption
of tissue and cell architecture among the methods tested, the
hydrogel disc method displays a remarkable similarity to the
results obtained using the conventional homogenization approach.
The manual spotted digestion extracts were evaluated: the number of
identified proteins was found similar to the number of identified
proteins from the other strategies considered in this study.
[0105] All the different on-tissue digestion strategies were
further validated using the LC-MS/MS data from the replicated
experiments. Thus, two parameters were considered for the
comparison. The first parameter was the number of protein groups,
which defines the minimum number of uniquely identified proteins
(when several possible proteins have highly similar sequences and
cannot be distinguished by the peptides identified in a given
experiment, these proteins are reported as a single group). The
second parameter used for the evaluation of all the different
digestion strategies was the number of distinct peptides: peptides
are considered distinct when they identify unique sequences of
amino acids. FIG. 10a displays the number of protein groups and of
distinct peptides which were obtained from the hydrogel digestion
experiments performed using both the microwave for 2 min and the
conventional oven for 4 hours: results were found to be very
similar for all the metrics compared. These results, along with
those of FIG. 9a confirm that accelerating the on-tissue digestion
using microwave radiation is reliable and it allows for an almost
identical population of proteins to be identified. Moreover, very
few differences were found when comparing the number of distinct
peptides; however, a statistically significant increase of the
peptides identified in the microwave digestion experiments was
observed. FIG. 10b illustrates the comparison between the
experiments performed on the 1 mm diameter rat brain thalamic
region. In this case, the number of protein groups was not
significantly changed among all the digestion strategies (FIG.
10b). Besides, considering the number of distinct peptides, the
manual spotting digestion strategy gave higher values. Thus,
probably the digestion efficiency of these approaches differed.
However, given that the same amount of trypsin was used in all the
methods, using the hydrogel disc as well as in the classic
on-tissue digestions, the difference may be due to the limited
volume of the hydrogel and also to the different incubation step
(microwave vs. oven). Furthermore, the Mann-Whitney U test (for the
hydrogel digestion experiments performed via microwave and oven)
and Kruskal-Wallis test (for all the digestions carried out using
the 1 mm rat brain tissue specimens) were applied to the number of
protein groups and the number of distinct peptides in order to find
possible statistical differences between groups. Significant
difference was found only for the number of distinct peptides
within the comparison microwave/oven (p<0.05).
CONCLUSIONS
[0106] The inventors have developed a method to significantly speed
up on-tissue protein digestion by applying microwave irradiation
for two minutes. This method can be used for histology-directed
analysis since it utilizes hydrogel discs fabricated at 1 mm
diameter that are precisely placed on defined regions, localizing
the digestion to a defined area of the tissue. The reliability and
reproducibility of the microwave assisted digestion has been
demonstrated by the comparison of the number of identified proteins
and other data from the LC-MS/MS experiments. This study
demonstrates that a rapid and reliable protein identification
strategy can be performed on a single tissue specimen while
preserving the inherent spatial information of the tissue. This is
of primary importance when the amount of material (tissue biopsy)
is often not enough for proteomics as well as for all the other
analysis that are usually carried out on a biopsy for clinical
investigations. In contrast, the conventional tissue homogenization
and digestion procedures are slower, more time consuming, and have
a significantly higher number of steps. This often results in the
need for a higher amount of starting material because of sample
loss resulting from handling of the tissue. Moving forward, the
hydrogel discs fabrication can be optimized for different
dimensions, according to tissue regions of interest. Future work
will also include optimization of the hydrogel methodology for
multiple enzymes experiment as well as for intact protein analyses.
Taken together, these results suggest the possible clinical utility
of histology-directed protein digestion approach.
[0107] The complete disclosure of all patents, patent applications,
and publications, and electronically available material cited
herein are incorporated by reference. The foregoing detailed
description and examples have been given for clarity of
understanding only. No unnecessary limitations are to be understood
therefrom. The invention is not limited to the exact details shown
and described, for variations obvious to one skilled in the art
will be included within the invention defined by the claims.
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